Blood Volume and Haemoglobin Mass in Relation to Fat-Free Mass and Aerobic Capacity in Elite Junior Rowers

Comment 1: Good introduction. The necessary background was provided without being verbose. The entire introduction logically leads to this statement. Considerable research exists specific to competitive rowing, and even rowing at the elite level. However, from what I can tell in common databases, there's a lack of research specific to hematology in elite junior rowers. These gaps in the research were also addressed by the authors in the introduction (lines 42-43, 48-50, 60-62). For a non-review paper, the manuscript is heavily referenced, and the citations are appropriately used throughout the manuscript. The tables and figures are appropriate, and they provide the reader with necessary detail not suitable for text. 

Response 1: Thank You for your positive comment.

Comment 2: I’m assuming that the bioimpedance measurement was taken before the GRXT, correct? Acute exercise affects the BIA measurement due to fluid shifts, changes in skin temperature, etc. Please include when BIA measurements were performed in the sequence of testing described in the study design (section 2.2).

Response 2: Thank you for this comment. Yes, the bioimpedance measurement was performed before the GRXT. We have clarified the order of procedures in the Methods section.

Comment 3: Was hydration monitored or quantified following the GRXT? I’m assuming that the athletes drank (ad libitum?) water or other fluids in the 2-hour interim between the GRXT and the blood draw. Some brief comment regarding how much this could have influenced the hematological measurements seems warranted in the discussion.

Response 3: Thank you for this important comment. Hydration was not formally monitored or quantified during the 2-hour period following the GRXT. However, participants were asked to drink water ad libitum during this interval. We agree that this should be clarified in the manuscript and acknowledged as a potential limitation. We have clarified it by adding comment at the end of 2.2 section: “During the 2-hour period following the GRXT, participants were asked to drink water ad libitum. Hydration intake during this interval was not standardized or quantified.”

Because fluid intake was not standardized or recorded, some influence on hematological variables related to plasma volume shifts cannot be excluded. However, total blood volume and hemoglobin mass were assessed two hours after the GRXT, which likely reduced the immediate acute effects of exercise-induced hemoconcentration.

So, we added this discussion to the limitations section: “Fourth, the hydration status was not formally monitored after the GRXT. Although participants were allowed to drink water ad libitum during the 2-hour recovery period, fluid intake was not quantified. Therefore, some residual influence of post-exercise changes in plasma volume on hematological measurements cannot be completely excluded. Nevertheless, the 2-hour interval between exercise cessation and blood-related measurements likely reduced the most immediate acute effects of exercise on these variables.”

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response 1: Thank You for your positive comment.

5. Additional clarifications

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Comment 1: Dear Authors: Your paper addresses an interesting topic—the physiology of exercise as it applies to high-performance rowing—with a specific focus on the relationship between aerobic capacity and hematological parameters.

The manuscript is well written. Below are some recommendations for further improving the manuscript, if applicable.

1.- Participants:

1.1. The study was conducted exclusively with Caucasian participants from the Lithuanian junior national team. This limits external validity, as the results may not be applicable to athletes from other ethnic groups or geographic regions with different genetic profiles or environmental conditions. Please justify the external validity, if you consider it applicable.

Response 1. Thank you for this important comment. We agree that the external validity of the present findings is limited by the relatively homogeneous sample, which consisted exclusively of Caucasian athletes from the Lithuanian junior national team. Therefore, our results should be interpreted primarily within the context of athletes with similar demographic and training backgrounds. We have revised the Discussion accordingly and now acknowledge that hematological variables and determinants of aerobic capacity may vary across ethnic groups and geographic environments. Further studies in more diverse athletic populations are needed to confirm the generalizability of these findings. We added this to study limitations section: ”Fifth, this study was conducted in a relatively homogeneous sample comprising only Caucasian athletes from the Lithuanian junior national team. Therefore, the findings are most directly applicable to athletes with similar demographic and training characteristics. Caution is warranted when extrapolating these results to other ethnic or geographic populations, as hematological variables related to oxygen transport, and potentially the determinants of aerobic capacity, may differ across populations due to genetic and environmental influences, such as ambient temperature or atmospheric pressure etc.”

Comment 2: 1.2 Although 39 athletes is a decent number for laboratory studies, it remains a small sample size for conducting complex statistical analyses or for dividing the data into subgroups (such as sex or rowing specialty) with high statistical power. Please justify the sample size.

Response 2: Thank you for this comment. We recognize that the relatively small sample size, especially in females, may have limited statistical power for some comparisons. A post hoc power analysis showed that the present sample size (25 males, 14 females) provided 80% power to detect an effect size of approximately d = 0.96 at α = 0.05. Thus, the study was adequately powered to detect large between-sex differences, but smaller differences, particularly in fat-free-mass-normalized variables, may have remained undetected. We have now acknowledged this as a limitation in the revised manuscript: ”the relatively small and unequal sample size between sexes, particularly the smaller female group, which may have reduced the robustness of the between-sex comparisons and limited the ability to detect smaller effects, especially in fat-free-mass-normalized variables.”

Comment 3: 1.3 There is an uneven representation between men (25) and women (14), which could affect the robustness of the comparisons made between the sexes.

Response 3: Thank you for this important comment. We agree that the unequal group sizes (25 men vs. 14 women) may reduce the robustness of between-sex comparisons, particularly for variables with smaller effect sizes. However, all available eligible athletes were included in the study, and the analyses were performed using the same procedures for both groups. In addition to p-values, we reported effect sizes to provide a more informative interpretation of the magnitude of between-sex differences. We have now acknowledged in the revised manuscript that the unequal sex distribution, together with the smaller female sample, should be considered when interpreting the comparisons: “the relatively small and unequal sample size between sexes, particularly the smaller female group, which may have reduced the robustness of the between-sex comparisons and limited the ability to detect smaller effects, especially in fat-free-mass-normalized variables.”

Comment 4: Furthermore, the participants were at the junior level: As athletes on the “junior national team,” the results are specific to adolescents or young adults still in development. It is possible that these parameters (especially blood volume and Hb mass) behave differently in elite senior rowers or recreational athletes. Please justify this choice.

Response 4: Thank you for this important comment. The focus on junior national-team athletes was intentional. We considered this cohort scientifically relevant in its own right, particularly because limiting the sample to one junior national-team program also reduced heterogeneity in training status and competitive level. We agree, however, that these findings should be interpreted specifically within the context of highly trained junior athletes and should not be directly generalized to elite senior rowers or recreational athletes. We have emphasized this in the beginning of 4.9 section: “The present findings are most directly applicable to highly trained junior athletes and should not be directly generalized to elite senior or recreational rowers.”

Comment 5: 1.5. In the women’s group, unless I am mistaken, I believe the phase of the menstrual cycle was not controlled for in the 14 rowers. This is a critical limitation, as hormonal fluctuations can significantly affect plasma volume and fluid retention. Please address this observation.

Response 5: Thank you for this important observation. We agree that menstrual cycle phase was not controlled or recorded in the female rowers. To our knowledge, fluctuations in estrogen and progesterone can influence fluid-regulatory mechanisms; however, the available literature suggests that overall plasma-volume changes across the menstrual cycle are generally small, and menstrual cycle phase does not appear to substantially alter ad libitum fluid balance or fluid retention. Accordingly, the lack of menstrual cycle control may have increased variability within the female group, but its effect on the main findings is likely limited. To avoid very long discussion, we skipped comprehensive analysis of this issue.

Comment 6: 1.6. Nor is it specified whether there was strict control of food intake, caffeine consumption, or hydration in the 24 hours prior to the test—factors that directly influence lactate and blood volume tests. Please justify this aspect.

Response 6: Thank you for this important comment. We agree that pre-test nutrition, caffeine consumption, alcohol intake, and hydration status may influence lactate- and blood-volume-related measurements. These factors were controlled before testing, but this was not described clearly enough in the original Methods section. We have now revised the manuscript to clarify it in 2.2 section: “Before testing, participants followed standardized pre-test instructions, including consumption of a standardized meal, abstention from alcohol, refraining from caffeine for at least 3 h, arriving fully hydrated, and avoiding high training volume or high-intensity exercise for at least 24 h before the laboratory visit.”

Comment 7: 1.7. Indicate the inclusion and exclusion criteria, if any.

Response 7: There were two main criteria of inclusion and this is stated in 2.1 section: “To be included in the study, the rowers were required to have at least five years of training experience and to be members of the national team for their age group.”

Comment 8: 2. Materials and Methods: There are some limitations.

2.1. Technological and Measurement Limitations. For body composition, the TANITA TBF–300 analyzer was used. Although practical, bioimpedance is less accurate than the “gold standard” (DEXA or hydrostatic weighing), as it can be affected by the athlete’s hydration status at the time of testing. Please explain.

Response 8: Thank you for this important comment. We agree that the use of the TANITA TBF-300 analyzer represents a methodological limitation. Bioelectrical impedance analysis estimates body composition indirectly from tissue conductivity and is sensitive to fluctuations in hydration status, which may affect the estimation of fat mass and fat-free mass. Compared with reference methods such as dual-energy X-ray absorptiometry (DXA) or hydrostatic weighing, this approach is less precise. We have added this limitation to the manuscript limitations section: “Sixth, body composition was measured using the TANITA TBF-300 analyzer, which applies bioelectrical impedance analysis. While this method is practical and suitable for routine assessment, it is less precise than gold-standard methods such as DXA or hydrostatic weighing. Moreover, because bioelectrical impedance is sensitive to hydration status and body fluid distribution, the obtained estimates of fat mass and fat-free mass may have been affected by acute physiological variations at the time of measurement. Consequently, the body composition results should be interpreted with caution.”

Comment 9: 2.2. Portable Gas Analyzer: The MetaMax 3B was used. Although the text mentions that it is reliable, portable systems sometimes exhibit slightly greater variability than stationary metabolic cart systems under controlled laboratory conditions. Please explain.

Response 9: Thank you for this important comment. We agree that portable gas analysis systems may show slightly greater variability or small systematic bias compared with stationary laboratory metabolic carts under highly controlled conditions. This limitation has been reported for the MetaMax 3B as well: although it has demonstrated acceptable reliability and stability, some studies have noted differences in VO₂ and VCO₂ compared with criterion systems, particularly at moderate-to-vigorous exercise intensities. However, the choice of the MetaMax 3B in the present study was deliberate, because its lightweight, wearable, breath-by-breath design is well suited to rowing ergometer testing and allows respiratory measurements to be obtained during a sport-specific movement pattern with minimal mechanical constraint. Thus, although a stationary metabolic cart may offer slightly higher analytical precision under laboratory conditions, the portable system provided important practical and ecological advantages for this protocol.

Comment 10: 3. Study Protocol and Design:
3.1. Measurements of blood volume (BV) and hemoglobin mass (Hb-mass) were taken just two hours after a maximal exercise test (GRXT). Although a recovery period is mentioned, the physiological stress of a maximal rowing test could temporarily influence plasma volume or fluid distribution. Nor is it mentioned whether the athletes were sufficiently hydrated after the exertion. These measurements should have been taken at complete rest and, of course, before the exercise tests. Please explain. 

Response 10: Thank you for this important comment. Hydration was not formally monitored or quantified during the 2-hour period following the GRXT. However, participants were asked to drink water ad libitum during this interval. We agree that this should be clarified in the manuscript and acknowledged as a potential limitation. We have clarified it by adding comment at the end of 2.2 section: “During the 2-hour period following the GRXT, participants were asked to drink water ad libitum. Hydration intake during this interval was not standardized or quantified.”

Because fluid intake was not standardized or recorded, some influence on hematological variables related to plasma volume shifts cannot be excluded. However, total blood volume and hemoglobin mass were assessed two hours after the GRXT, which likely reduced the immediate acute effects of exercise-induced hemoconcentration.

So, we added this discussion to the limitations section: “Fourth, the hydration status was not formally monitored after the GRXT. Although participants were allowed to drink water ad libitum during the 2-hour recovery period, fluid intake was not quantified. Therefore, some residual influence of post-exercise changes in plasma volume on hematological measurements cannot be completely excluded. Nevertheless, the 2-hour interval between exercise cessation and blood-related measurements likely reduced the most immediate acute effects of exercise on these variables.”

Comment 11: 3.2. The design is a single-time cross-sectional study. (“Participants had to visit the laboratory once.”) This provides a “snapshot” of the moment but does not allow for observing the variability of the parameters over the course of a competitive season or across different training states. Please explain why longitudinal measurements were not taken throughout the season.

Response 11: Thank you for this comment. We agree that a longitudinal design would provide valuable information regarding seasonal and training-state variability. Nevertheless, the purpose of the present study was to examine the selected parameters at a single, standardized time point rather than to track their changes throughout the season. For this reason, a cross-sectional single-visit design was chosen as the most appropriate approach for the specific aims of this manuscript. We acknowledge that repeated measurements across the season may yield additional insights; however, such analyses fall outside the scope of the present study and will be addressed separately.

Taking this into account we have modified first study limitation: “First, the present study used a single-time cross-sectional design; therefore, the results represent only a snapshot and do not reflect possible seasonal or training-state variability. However, this approach was appropriate for the specific aims of the study, which did not require longitudinal monitoring. Future studies should examine these parameters repeatedly across different phases of the training and competitive season.”

Comment 12: 4. Statistical Analysis: 
4.1. The statistical analysis described is appropriate and standard for this type of sports physiology study. It is correct to perform a normality test before deciding to use ANOVA or Pearson’s correlations, since these are parametric tests that assume the data are normally distributed. The use of Effect Size (Cohen’s d) is appropriate. In sports science, the “p” value only tells us if there is a difference, but the effect size (d) indicates how large and relevant that difference is in practice. 

Response 12: Thank you for your positive assessment.

Comment 13: 4.2. Using SPSS v.27 is appropriate, as it is a contemporary and robust version.

Response 13: Thank you for your positive assessment.

Comment 14: 5. Nomenclature errors: On lines 141–142, “Shapiro-Wilcoxon test” is written. I believe there has been a mix-up. You are likely referring to the Shapiro-Wilk test, which is the standard test for assessing normality in small samples (n < 50). The Wilcoxon test is a nonparametric test (an alternative to Student’s t-test). Mixing the two names is a technical error in the manuscript, although it is understood that your intention was to verify whether the data followed a normal distribution.

Response 14: Thank you for this careful observation. We fully agree that the term “Shapiro-Wilcoxon test” was incorrect. The appropriate test used in our analysis was the Shapiro–Wilk test, which is commonly applied to assess normality, particularly in small samples. The manuscript has been corrected accordingly.

Comment 15: 6. The results are well presented and easy to understand. 
It is not essential, but I believe that the sample data—especially for coaches—would be more informative if Table 1 had included the best times in the 2000-meter race for both men and women. We already know that times in this event vary depending on weather conditions and other external variables and are highly dependent on age, but in a junior national team, it is easy to achieve a better time in this event. Additionally, the following tables/figures could indicate the relationship between best 2000 m time and kg or FFM.

Response 15: Thank you for this valuable suggestion. In response, we have added the athletes’ best 2000 m competitive times for both male and female rowers to Table 1, as we agree that these data may be informative for coaches and readers. However, we did not calculate correlations between competitive performance and anthropometric, aerobic, or hematological variables because the athletes competed in different boat classes. Under these conditions, race performance cannot be considered directly comparable, since it is affected by boat category, crew-related factors, pacing strategy, and external race conditions. Therefore, such analyses would not provide a sufficiently valid interpretation in the context of the present study. To examine these relationships properly, standardized individual performance testing would be required, for example by using a 2000 m rowing ergometer test or single-boat assessment. This type of analysis was beyond the scope of the current study.

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response 1: Thank you for your positive assessment.

5. Additional clarifications

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Comment 1: In the introduction:
Please expand on the topic of training experience and its impact on the mentioned load parameters in rowers. Are there any differences based on training level, performance level, and competitive experience?

Response 1: Thank you for this valuable comment. We agree that training experience, performance level, and competitive experience are important factors when interpreting physiological characteristics in rowers. We have therefore expanded the third paragraph of the Introduction: “Since higher fat–free mass (FFM) and lower fat mass (FM) are linked with better rowing performance, body mass (BM) and composition should also be considered when assessing BV, Hb–mass, and V̇O2max. The higher absolute V̇O2max, BV and Hb–mass values in rowers may be due to their higher BM and FFM than those of other athletes [14]. In addition, these physiological characteristics are influenced by training experience, performance level, and competitive background. Reference values for V̇O2max differ across junior, U23, and senior elite rowers, and longitudinal data in adolescent rowers show further increases in aerobic and anaerobic power with continued training and maturation. Similarly, hematological adaptations are related to endurance training status, and changes in training load may influence plasma volume, blood volume, and exercise capacity. Therefore, training age and competitive level should be considered when interpreting physiological profiles in junior rowers. [5, 15]. There are no data concerning elite junior female rowers.”

Comment 2: Materials and Methods:
In this section, please attach a graph illustrating the course of the research experiment, including the selection and selection of the research group, the inclusion and exclusion criteria, and all activities undertaken throughout the experiment. - In this section, it's also worth adding an illustration or photograph of the athlete performing the test.

Response 2: Thank you for the suggestion. We added the study protocol flowchart to the manuscript. Since the study is already completed and we had not taken any pictures during the graded rowing exercise test, unfortunately, we cannot add any photographs.

Comment 3: Please indicate the criteria used to select the load for the test, and whether it took into account the level of training, body weight, or athletic ability. Was the only differentiating factor the gender of the subjects?

Response 3: Thank you for this comment. The test load was differentiated primarily by sex, which also reflects expected differences in rowing performance capacity. Because the participants constituted a relatively homogeneous group of elite junior rowers, the protocol was not further individualized by body mass or training level. The workload increased every 30 s, resulting in a rapidly progressive test in which small differences in the starting load would have had only a limited influence on test duration and on the peak responses obtained. Therefore, a standardized sex-specific protocol was considered appropriate for the aims of the present study. We have clarified it more in 2.3.3. section by adding: “The initial workload was selected according to participant sex, reflecting known sex-related differences in rowing performance capacity. No additional adjustment for body mass or training level was applied because all participants were elite junior rowers with a relatively homogeneous training background.”

Comment 4: Please also describe in this section the method used to select the study participants. Were all athletes who finished 10th or lower at the European Championships recruited?

Response 4: Thank you for this comment. We have clarified the participant selection procedure in the revised manuscript. The athletes were recruited from the Lithuanian junior rowing national team using a purposive sampling approach. All eligible athletes available during the study period were invited to participate. Eligibility required at least five years of training experience, membership in the national team for the relevant age group, and absence of acute illness or injury at the time of testing. So, we revised 2.1. section: “Thirty-nine competitive academic rowers (twenty-five males and fourteen females) from the Lithuanian junior rowing national team were recruited for the study using a purposive sampling approach. All eligible athletes who were available during the study period were invited to participate. To be included in the study, the rowers were required to have at least five years of training experience, to be members of the national team for their age group, and to be free from acute illness or injury at the time of testing. The participants ranked among the top ten in their respective age groups at the European Championships, which reflects the high competitive level of the sample. All participants were Caucasian. The descriptive characteristics of the participants are presented in Table 1.”

Comment 5: Please also provide information on whether any athletes withdrew or refused to participate in the study, either before or during the ergometer test.

Response 5: Thank you for this comment. No athletes withdrew from the study or refused to participate, either before or during the ergometer test. This information has now been added to the revised manuscript.

Comment 6: Results:
The results presented in this section are very good and do not require correction. However, it is worth considering adding correlation results between sporting level, competitive experience, and the parameters studied, which would certainly be a valuable observation in this study.

Response 6: Thank you for this valuable suggestion. In response, we have added the athletes’ best 2000 m competitive times for both male and female rowers to Table 1, as we agree that these data may be informative for coaches and readers. However, we did not calculate correlations between competitive performance and anthropometric, aerobic, or hematological variables because the athletes competed in different boat classes. Under these conditions, race performance cannot be considered directly comparable, since it is affected by boat category, crew-related factors, pacing strategy, and external race conditions. Therefore, such analyses would not provide a sufficiently valid interpretation in the context of the present study. To examine these relationships properly, standardized individual performance testing would be required, for example by using a 2000 m rowing ergometer test or single-boat assessment. This type of analysis was beyond the scope of the current study.

Comment 7: Discussion:

In the discussion, it is worth referring to the competitive experience and the results obtained, as well as the sporting level, and explaining whether the parameters studied are significant with higher sporting level.

Response 7: Thank you reviewer for this suggestion. In the revised methods and discussion sections, we added the athletes’ mean 2000 m competitive times to better characterize the general sporting level of the cohort. At the same time, we clarified that these times were not suitable for correlation analysis with aerobic capacity or blood variables, because rowing performance in competition is substantially affected by boat class and environmental conditions, including weather. Therefore, the relationship between sporting level and the studied parameters is discussed only theoretically and with reference to previous literature. We added some additional comments about this in 4.9. section: “The mean 2000 m competitive times of the studied athletes (377.8 ± 8.2 s in males and 434.8 ± 11.5 s in females) indicate the general performance level of the cohort. However, these results should be interpreted with caution, as competitive times in rowing are influenced not only by physiological capacity but also by boat class, environmental conditions, weather, and race context. Therefore, in the present study, competitive performance data were used only to describe the sporting level of the athletes and not for formal correlation analysis with aerobic capacity or hematological variables. Consequently, any relationship between higher sporting level and the studied physiological parameters can only be discussed theoretically and in light of previous literature. The present findings are most directly applicable to highly trained junior athletes and should not be directly generalized to elite senior or recreational rowers.”

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response 1: Thank you for your positive assessment.

5. Additional clarifications

–